Monoclonal antibodies useful in deodorizing skin and antibody fragments thereof

ABSTRACT

The present invention is concerned with a monoclonal antibody or antigen-binding fragment of a monoclonal antibody produced by a hybridoma formed by fusion of cells from mouse myeloma cells and spleen cells wherein the monoclonal antibody or fragment thereof reacts with the carrier or transfer protein of a bacterial cell. The bacterial cell may be coryneform bacteria or bacteria of the genus Staphylococcus.

This application is a divisional application of 07/360,154, filed Jun.1, 1989 now U.S. Pat. No. 5,069,896.

BACKGROUND OF THE INVENTION

The present invention relates to a monoclonal antibody (McAb) producedby a hybridoma cell line. The McAb has specificity to transport proteinsof bacterial cells and results in the inhibition of the formation ofaxillary malodor.

Human body odor is caused by bacteria that normally inhabit the skin.These bacteria may rely on some components of perspiration which serveas nutrients for the growth of the bacteria. Based on the amount ofperspiration formed, the axillary area is one of the primary areas ofconcentration of bacteria on the human body. The secretions inperspiration may serve as nutrients for bacterial growth or as precursorcompounds for bacterial metabolic pathways leading to malodor formation.

Various approaches have been taken to solve the problem of axillarymalodor. One approach has been the use of antiperspirants.Antiperspirants may prevent the formation of odor by inhibitingperspiration, thereby depriving the bacterial metabolic pathways leadingto malodor formation of the necessary substrates, or by exerting adirect antimicrobial effect on the bacteria present in the axilla.Another approach has been the use of deodorants which attempt to maskthe odor produced. A third approach has been the use of germicides whichkill or inhibit the reproduction of bacteria.

A number of prior art publications have suggested using antibodies whichreact with and thereby kill certain species of bacteria. It is knownthat the body produces different types of antibodies which function indifferent environments.

There are five known classes of antibodies including IgG, IgM, IgA, IgEand IgD. Four of the antibody classes, namely IgG, IgM, IgE and IgD, arereferred to as humoral antibodies because they naturally occur in theblood and function in those parts of the body that come in direct orindirect contact with the blood. Humoral antibodies can also be found inthe tissue fluids of the body. The tissue fluids receive the humoralantibodies of the blood by diffusion of the antibodies from the bloodinto the surrounding tissue fluids by a process known as transudation.The IgA antibody is referred to as a secretory antibody because it isfound in the fluids secreted by the epithelial cells which line thesurfaces of hollow body organs. The IgA antibody functions as a barrierby protecting the surface of the gastrointestinal tract from infectionby bacteria and viruses and by preventing the absorption of toxins andpoisons by the gastrointestinal epithelium.

Milk is a logical choice for the production of a deodorant antibodysince it contains the same classes of antibody found in secretions ofmammalian sebaceous glands. The only exception is the milk of dairy cowscontains principally IgG antibody.

An example of antibody-containing milk is disclosed in U.S. Pat. No.3,376,198. The antibody-containing milk is effective in providingantibodies which counteract a number of different bacteria and virusesdepending upon the antigen administered to a cow. U.S. Pat. No.3,128,230 also illustrates an antibody-containing milk generated morespecifically against the bacterial species Escherichia coli,Streptococcus viridans and Diplococcus pneumoniae. U.S. Pat. No.3,907,987 discloses an antibody-containing milk which is effectiveagainst microorganisms responsible for enteric diseases.

European Patent Application No. 0,127,712 suggests using a non-specificantibody preparation for milk directed against a large number ofbacterial species associated with human skin as a deodorant. Thisantibody was prepared without a specific understanding of themalodor-causing bacterial species which inhabit human skin.

Antibodies can be developed against specific bacterial species or evenspecific enzyme systems within a bacterial cell (Brit J. Dermatol.116:805-812, 1978). This specificity can be achieved by developing aMcAb directed against a particular bacterial system associated with themalodor-forming pathway.

The prior art has not heretofore suggested or recognized the possibilityof producing a McAb to the cellular transport protein of bacteria whichis responsible for delivering the malodor precursor compound into thebacterial cell.

SUMMARY OF THE INVENTION

The present invention is a method of preventing the formation ofaxillary malodor by applying to a human a composition comprisingantibodies directed against the transport protein of bacterial cellsinvolved in the production of malodor.

Contrary to any prior art processes heretofore recognized, by inhibitingthe transport of selected compounds into the bacterial cell, malodorwould be prevented, leaving the bacterial cell intact and viable.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes the discovery that malodor can bedecreased or eliminated using the McAb produced by a hybridoma cell linewherein the antibody has specificity against a transport protein used totransfer certain compounds into the cell.

The skin areas producing the greatest perspiration also have thegreatest bacterial populations However, not all of the bacteria whichare present on the skin cause malodor. The malodor-causing bacteria arethose which can absorb and convert specific precursor compounds into oneor more malodorous compounds using their cellular metabolism. Forinstance, species of bacteria which produce malodor from secretions inperspiration include Staphylococcus naemolyticus and the coryneformbacteria.

The identification of bacteria as malodor-producers involves a screeningmethod for determining the presence or absence of a volatile malodorcompound. One such method involves a) adding 100 microliters of amalodor-producing cell suspension to a vial which contains concentratedand dried malodor precursor compound, b) incubating the mixture forapproximately 30 minutes at 37° C., c) adding 200 microliters ofchloroform then capping the vials, d) allowing the vials to stand for atleast 15 minutes at room temperature, e) removing about 50 microlitersof the chloroform layer and applying the chloroform layer to a strip ofbibulous paper, and f) sniffing the paper for the presence of malodorafter the chloroform has evaporated.

The malodor-producing bacteria are unique in that they possess anintracellular malodor-forming enzyme. It is a metalloenzyme which actsas an amino acid lyase and its activity depends upon the presence ofpyridoxal phosphate as a co-factor. Upon storage at 4° C., thesebacteria lose the ability to produce malodor; however, after harvest andlysis of the cells, an active malodor-forming enzyme can be recovered.While not wishing to be bound by any particular theory, this suggeststhat a labile transport system for the malodor precursor compound isrequired for the transfer of precursor compounds into the cell whereuponit serves as a substrate for the malodor-forming enzyme.

Ions, sugars and amino acids are transported into bacteria using eithergroup translocation, facilitated diffusion, simple diffusion or activetransport. Amino acids are usually transported by active transportmechanisms which require the expenditure of cellular energy to transporta compound up a chemical gradient. It has been shown that structurallyrelated amino acids use the same active transport carrier. There isevidence that the malodor precursor compound, a sulfur-containing aminoacid, uses the same active transport carrier as L-cysteine. A McAbspecific for the precursor carrier protein blocks the formation ofmalodor by preventing precursor uptake by the cell.

Recently, it has been demonstrated that a McAb can be produced in miceto react with an extracellular toxin produced by the fungus Fusarium asnoted in Appl. Environ. Microbiol., 54:2959-2963, 1988. The stepsemployed in McAb development against a variety of specific targetsubstrates have some general procedures in common. The first stepinvolves the development of a specific antigenic material which willinduce an immunologic response from antibody-generating cells in vivo.These immunocytes are then recovered and fused with an appropriateimmortalizing cell line such as mouse myeloma cells. Subsequent to thisfusion, the resulting hybridoma cells are grown under conditions whichallow for production of the specific desired antibody. Hybridomacolonies are recovered and screened for this ability to produce theantibody, then the most enzyme-linked immunosorbent assay-positivehybridoma lines are cloned. Once these lines are cloned, known methodscan be employed to maximize the production of the McAb.

The McAb-containing material can be applied to the skin in a variety ofways to accomplish the intended object of deodorizing the skin. TheMcAb-containing substance itself may be applied directly to the skin,preferably the axilla, or it may be applied as the active ingredient ina composition comprising the McAb and an inert vehicle. Suitablevehicles include gels, liquid sprays (pumps and aerosols), creams,liquids, lotions, oils, ointments, solid stick applicators and the like.The most preferred compositions for application to the underarms aresolid sticks, roll-on emulsions, creams and lotions.

In all formulations, it is important that the McAb be compatible withthe ingredients of the topical skin vehicle, that is, the McAb mustmaintain its viability in the formulations selected. For example,nothing that may denature the McAb-containing material should be presentin the formulation. In general, there should be less than 10% alcoholand less than 1% denaturing detergents The preferred propellant in theformulation is CO₂ :N₂.

Any of the ingredients commonly employed in the manufacture ofcompositions applied topically to the skin, and which have no adverseeffect on the active ingredient, may be used. The use of an FAb, theantigen-binding fragment of the McAb, may be more stable than the entireMcAb and thus more efficacious in these compositions.

There are a variety of methods known in the art for producing FAb.Methods for producing FAb fragments directly from cell culturesupernatants include digestion of the antibody with pepsin at pH 3.5 to4.0. The pepsin at a concentration of 25 ug/ml in 0.1M citrate buffer,pH 3.5, can digest IgG antibody incorporated at 1 to 2 mg/ml. Similardigestion systems using trypsin having been shown to produce FAbfragments from IgM antibodies in the presence of a reducing agent.Purification of FAb fragments is completed by dialysis followed bycolumn chromatography. Antigen-binding capacity has been found to remainassociated with both heavy-chain and light-chain binding sitesassociated with the resultant FAb fragments.

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat these examples are intended only to be illustrative without servingas a limitation on the scope of the present invention.

EXAMPLE 1 Preparation of an Antigen for the Development of anAnti-Malodor Antibody

Malodor-producing Staphylococcus and coryneform bacteria were isolatedfrom the axillae of several individuals by washing the axilla with 2.0ml of sterile saline, diluting this wash serially in saline, and platingthe cell suspension on letheen agar plates. The inoculated plates wereincubated at 37° C. for 24 hours followed by an additional incubation at25° C. for 24 hours. Large yellow colonies were examined microscopicallyand biochemically and were typed as Staphylococcus haemolyticus, whichwhen screened for the ability to produce malodor as described above werefound to be malodor-positive. Similarly, small translucent colonies weretyped as bacteria of the coryneform group and were alsomalodor-positive.

A 100 ml culture of the Staphylococcus was grown in Tryptic Soy Broth,harvested, and re-suspended in 50 mM phosphate buffer, pH 6.8. The cellswere lysed using a sonication method, and cell wall and cell membranefragments were separated from the soluble materials by centrifugation.

The cell wall/membrane fraction was diluted to 38.1 microgramsprotein/ml in 0.1M carbonate buffer, pH 9.6. This preparation was usedas the antigen in an immunization schedule in which Balb/c mice werechallenged and boosted over a 12-week period.

EXAMPLE 2 Immunization of Mice for Raising an Anti-Malodor Antibody

In vivo immunization of Balb/c mice was conducted using the antigendescribed above. Each mouse received 100 micrograms of antigen persubcutaneous inoculation. Sera collection included one pre-inoculationbleed and three test bleeds. Each test bleed was performed 10 days aftera boost inoculation. The pre-bleed and three test bleeds wereindividually screened for potential anti-malodor antibody activity bytreating the washed-cell suspension, containing on the order of 10⁹cells/ml, with an equal volume of test serum. Each mixture was incubatedfor one hour at room temperature then screened for the production of thevolatile malodor compound according to the method described above.

Activity of the sera was simultaneously screened against the specificantigenic preparation by ELISA. The mouse having the highest titer ofanti-Staphylococcus cell wall/membrane antibody was sacrificed forsubsequent recovery of antibody-producing splenocytes. It was noted thatthe serum from this mouse also had anti-malodor antibody activityagainst both Staphylococcus and coryneform bacteria as determined by themalodor assay.

EXAMPLE 3 Development of a Hybridoma Cell Line for the Production of anAnti-Transport Monoclonal Antibody

The mouse having the highest serum titer of desired antibody, asdetermined by the ELISA and malodor assays, was sacrificed and thespleen removed under aseptic conditions. A suspension of splenocytes of2.2×10⁷ cells/ml was prepared. A suspension of cells of 6.2×10⁶ /ml froma myeloma cell line was also prepared. The splenocytes and myeloma cellswere combined in a ratio (spleen:myeloma) of 4:1. The cells were treatedand recovered by centrifugation in Iscove's Modified Dulbecco's Medium(IMDM) with hypoxanthine-adenine-thymidine (HAT) in the presence of 50%(w/v) polyethylene glycol. Following this fusion treatment, the cellswere recovered by centrifugation and resuspended in 33.0 ml of IMDM withHAT for every 1.6×10⁸ cells. The suspension was dispersed dropwise intoa total of 600 wells in microwell plates and incubated for seven days at37° C. in a dry CO₂ (5%) incubator. The wells which had 30-50% confluentgrowth of hybridoma cells were screened by ELISA and by the malodorassay. Each hybridoma line (corresponding to a well) which wasassociated with both a positive ELISA response and a positive activityagainst malodor production was chosen for subsequent cloning.

The desired hybrid cells were diluted serially with IMDM to obtain afinal concentration of 100 cells in 30 ml IMDM and 10% fetal bovineserum. Spleen cells were added to give a concentration of 3-5×10⁶ feedercells/ml. The suspension was dispersed into microwell plates andincubated at 37° C. in a CO₂ (5%) incubator. Following growth, theclones were again assayed for a positive ELISA response and activityagainst malodor production. Lines having the highest titers were chosenfor a second cloning and tested again.

All hybridoma cell lines having been cloned at least one time andshowing significant activity by ELISA and malodor screens were preservedby controlled rate freezing in IMDM. The cell lines were stored incryopreservation tubes in liquid nitrogen.

EXAMPLE 4 In Vitro Production of Anti-Transport Monoclonal Antibody andQuantitation of its Activity

Individual hybridoma cell lines were recovered from frozen stocks andexpanded in WRC 935 serum-free basal medium manufactured by W.R. Grace &Co. supplemented with insulin, transferrin and albumin. At confluency,cells were passed by a scraping technique and were transferred to newflasks containing fresh medium. The antibody-containing medium wascollected semi-weekly and the cells were introduced to fresh medium. Thecollected medium was stored at 4° C. until procedures for thequantitation of the antibody were performed.

The collections of antibody-containing media were pooled thenconcentrated to a 50 ml volume using an Amicon stirring ultrafiltrationcell and membrane with a molecular weight cut-off of 30,000 daltons. Theantibody-containing fraction was further concentrated by lyophilization.Lyophilized product was dissolved in distilled, deionized water.Quantitation was performed using both the ELISA and malodor assaytechniques. Application of the anti-transport McAb to anSDS-polyacrylamide electrophoresis gel followed by a Western blotindicated that the McAb reacts with a single protein from theStaphylococcus cell wall/membrane fraction having a molecular weight of50,000 daltons.

In vitro malodor assays on McAb-treated malodor-producing Staphylococcuscells showed that between 7 and 35 nanograms of the McAb were requiredto block precursor transport in an order of 10⁸ bacteria. This exceedsthe concentration of malodor-producing cells present in the axilla.Therefore, it is expected that the McAb is an effective anti-transportagent at low concentrations.

A human malodor neutralization study was conducted and the McAbformulations were found to be effective.

EXAMPLE 5

A deodorant stick formulation may be prepared as follows:

    ______________________________________                                        Compound Name         %                                                       ______________________________________                                        Methyl gluceth-20 distearate                                                                        10                                                      Propylene Glycol      72                                                      Sodium stearate C-1   6                                                       Fragrance             0.1                                                     0.2%, Active ingredient in water,                                                                   11.9                                                    neutral pH                                                                    ______________________________________                                    

EXAMPLE 6

A deodorant roll-on emulsion may be prepared as follows:

    ______________________________________                                        Compound Name           %                                                     ______________________________________                                        Hydrogenated palm oil glycerides and                                                                  1                                                     sodium cetyl sulfate (Lamecreme CSM)                                          Steareth-7 (Lamecreme SA-7)                                                                           1                                                     Octyldodecanol          4                                                     Glyceryl laurate (Monomuls 90-1 12)                                                                   2                                                     Octyl palmitate         4                                                     Dimethicone             1                                                     Propylparaben           0.1                                                   Methylparaben           0.2                                                   Imidazolidinyl urea     0.3                                                   Glycerin                5                                                     Allantoin               0.5                                                   PEG-35 lanolin (Lamecerin 50-80)                                                                      0.5                                                   Fragrance               0.3                                                   0.3%, Active ingredient in water,                                                                     79.6                                                  neutral pH                                                                    ______________________________________                                    

EXAMPLE 7

An antiperspirant and deodorant roll-on emulsion may be prepared asfollows:

    ______________________________________                                        Compound Name            %                                                    ______________________________________                                        PPG-15 steatyl ether (Arlamol E)                                                                       4                                                    Steareth-21 (Brij 721)   0.6                                                  Steareth-2               2.6                                                  Aluminumzirconium pentachlorohydrate,                                                                  32                                                   10:1, 25% solution                                                            Fragrance                0.1                                                  0.4%, Active ingredient in water,                                                                      60.7                                                 neutral pH                                                                    ______________________________________                                    

EXAMPLE 8

An antiperspirant and deodorant stick formulation may be prepared asfollows:

    ______________________________________                                        Compound Name             %                                                   ______________________________________                                        Aluminum chlorohydrate (Wickenol                                                                        16                                                  CPS-331                                                                       Polypropylene glycol      30                                                  Sorbitol, 70%             3                                                   Sodium stearate C-1       5                                                   Sodium ceteth-13 carboxylate (Sandopan KST)                                                             3                                                   Stearyl alcohol Aldol 62 Flake)                                                                         1                                                   Cyclomethicone (Volatile Silicon 7158)                                                                  15                                                  Fragrance                 0.1                                                 0.1%, Active ingredient in water                                                                        26.9                                                neutral pH                                                                    ______________________________________                                    

EXAMPLE 9

An antiperspirant and deodorant cream formulation may be prepared asfollows:

    ______________________________________                                        Compound Name          %                                                      ______________________________________                                        Condensate of ethylene oxide with a                                                                  5                                                      hydrophobic base formed by                                                    condensing propylene oxide with                                               propylene glycol (Pluronic F-68)                                              Cetyl trimethyl ammonium bromide                                                                     0.5                                                    Cetyl alcohol          1                                                      Glyceryl monostearate  13                                                     Spermaceti wax         4                                                      Glycerine              3                                                      Polyoxyalkylene propylene glycol                                                                     0.5                                                    monostearate                                                                  Polyoxyalkylene stearate                                                                             0.5                                                    Ethanol                2                                                      Fragrance              0.1                                                    0.2%, Active ingredient in water,                                                                    70.4                                                   neutral pH                                                                    ______________________________________                                    

EXAMPLE 10

An antiperspirant and deodorant lotion formulation may be prepared asfollows:

    ______________________________________                                        Compound Name          %                                                      ______________________________________                                        Condensate of ethylene oxide with a                                                                  10                                                     hydrophobic base formed by                                                    condensing propylene oxide with                                               propylene glycol (Pluronic F-68)                                              8-Hydroxyquinoline sulfate                                                                           0.8                                                    Ethanol                2                                                      Veegum                 3.5                                                    Mineral oil            6                                                      Stearyl alcohol        1.5                                                    Polyoxyalkylene propylene glycol                                                                     0.8                                                    monostearate                                                                  Polyoxyalkylene stearate                                                                             0.8                                                    Fragrance              0.1                                                    0.2%, Active ingredient in water,                                                                    74.5                                                   neutral pH                                                                    ______________________________________                                    

EXAMPLE 11

An aerosol deodorant formulation may be prepared as follows:

    ______________________________________                                        Compound Name         %                                                       ______________________________________                                        Dioctyladipate        10                                                      Quaternium 18 hectorite                                                                             1                                                       Dioctyl succinate     10                                                      SDA 40 ethanol, anhydrous                                                                           1                                                       Wheat germ glycerides 0.1                                                     Fragrance             0.1                                                     0.2%, Active ingredient in water,                                                                   1                                                       neutral pH                                                                    CO.sub.2 :N.sub.2 Propellent                                                                        76.8                                                    ______________________________________                                    

EXAMPLE 12

An aerosol antiperspirant and deodorant formulation may be prepared asfollows:

    ______________________________________                                        Compound Name          %                                                      ______________________________________                                        Isopropyl myristate    13.4                                                   Aluminum chlorhydrate  10                                                     Quaternium-18 hectorite (Bentone 38)                                                                 0.8                                                    SDA 40 anhydrous       0.8                                                    Fragrance              0.1                                                    0.2%, Active ingredient in water,                                                                    1                                                      neutral pH                                                                    CO.sub.2 :N.sub.2 Propellent                                                                         26.9                                                   ______________________________________                                    

What is claimed is:
 1. A murine monoclonal antibody produced by ahybridoma formed by fusion of cells from mouse myeloma cells and spleencells, wherein said antibody specifically binds with the 50,000 daltoncarrier or transfer proteins of a malodor producing bacterial cell toprevent malodor formation, said bacterial cell being a coryneformbacteria or of the genus Staphylococcus.
 2. The monoclonal antibodyaccording to claim 1, wherein said antibody reacts with the carrier ortransfer protein to block the uptake of precursor compounds whichproduce malodor into the bacterial cell.
 3. The monoclonal antibodyaccording to claim 1 wherein the bacterial cell is of the genusStaphylococcus.
 4. The monoclonal antibody according to claim 2 whereinthe bacterial cells is coryneform bacteria.
 5. The monoclonal antibodyaccording to claim 1, wherein said monoclonal antibody is the IgG type.6. The monoclonal antibody according to claim 1 wherein said monoclonalantibody is the IgM type.
 7. The monoclonal antibody according to claim1 wherein the carrier or transport protein is found in the cell wall ofmalodor-producing skin bacteria.
 8. An antigen-binding fragment of amonoclonal antibody according to claim
 1. 9. The fragment according toclaim 8 wherein the bacteria are of the genus Staphylococcus.
 10. Thefragment according to claim 8 wherein the bacterial are coryneformbacteria.